Cyanogen Bromide Cleavage and Partial Amino Acid Sequ ence of Porcine Growth Hormone*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 245, No. 13, Issue of July 1, pp , 19iO Printed in U.S.A. Cyanogen Bromide Cleavage and Partial Amino Acid Sequ ence of Porcine Growth Hormone* JOHN B. MILLS, STEWART C. HOWARD, SYLVIA SCAPA, AND ALFRED E. WILHELMI$ From the Department of Biochemistry, Emory University, Atlanta, Georgia (Received for publication, December 5, 1969) 5OSZ?FZ SUMMARY When samples containing homoserine and homoserine lactone Porcine growth hormone has been cleaved with cyanogen were analyzed, the short column and the first part of the long bromide and the resulting fragments separated by gel filtracolumn (until glutamic acid emerged) were run at 5 instead of tion. The amino acid sequences of the aminoterminal the usua1 55 * Under these conditions homoserine lactone was tetrapeptide (Fragment D), the carboxyterminal dodecaresolved from ammonia, and homoserine was resolved from peptide (Fragment C), and another polypeptide of 55 residues glutamic acid. (Fragment B) are reported. Preliminary chemical evidence Samples containing Saminoethylcysteine, after being hydrolyzed and dried, were treated with.1 ml of 2 N NaOH and and comparison with the primary structure of human growth hormone indicate that Fragment B is adjacent to Fragment C dried again before being dissolved in sample buffer for analysis. in native porcine growth hormone. There is at least 67% homology between pig and human growth hormones in the carboxyterminal third of the molecules. In view of interest in the species differences in pituitary growth hormone, and the reports on the primary structures of human growth hormone (I) and of bovine growth hormone (2, 3), we are reporting data on the structure of porcine growth hormone. The latter has been cleaved at its 3 methionine residues with cyanogen bromide, the resulting fragments separated by gel filtration, and the amino acid sequence of three of the fragments determined. EXPERIMENTAL PROCEDURE Xource of Porcine Growth HormonePorcine growth hormone was prepared in this laboratory by the method of Chen, Wilhelmi, and Howard (4). All of the hormone used in structural work was eluted from DEAEcellulose at.375 M Trisformate, ph 8.. The several porcine growth hormone preparations used in this study have had mean biological potencies in the range of 1.7 to 2.1 i.u. per mg, measured by the weight gain test in hypophysectomized rats (5). Amino Acid AnalysisAmino acid analyses were performed on a Spinco model 12B amino acid analyzer by the method of Benson and Patterson (6). Samples of protein were hydrolyzed in 6 N HCl under reduced pressure (oil pump) at 15 for 24, 48, and 72 hours. The samples of fragments and peptides obtained were hydrolyzed under the same conditions for 18 to 24 hours. * This research was supported by United States Public Health Service Grants AM 3598 and HD Publication 945, Division of Basic Health Sciences, Emory University. $ To whom reprint requests should be addressed. FRACTION NUMBER FIG. 1. Separation on Sephadex G75 of the cyanogen bromide fragments from porcine growth hormone. A column, 3.7 X 17 cm, was eluted in 2y formic acid at a flow rate of 22.5 ml per hour. The product mixture from 1 g of reduced and Saminoethylated hormone was applied to the column. This treatment reduced the content of ammonia to improve the resolution of lysine, and, at the same time, any homoserine lactone present in the sample was almost completely converted to homoserine. To resolve Xaminoethylcysteine from lysine, a 14cm column of Aminex Q 15s resin was run at 5 with.55 M sodiumcitrate buffer, ph 3.96, for 35 min, followed by.55 M sodiumcitrate buffer, ph The basic amino acids emerged from the short column in the following order: Saminoethylcysteine, lysine, ammonia, histidine, homoserine lactone, arginine. Analysis for tryptophan was carried out by reaction with Nbromosuccinimide (7, 8). Reduction and Carboxymethylation or AminoethylationTotal reduction of protein disulfides and Scarboxymethylation was carried out as described by Crestfield, Moore, and Stein (9). 347

2 348 Partial of Porcine Growth Hormone Vol. 245, No. 13 Amino acid composition of porcine growth hormone and its cyanogen bromide fragments Values are expressed as residues per mole. The numbers in degradation (see Experimental Procedure or Reference 15) for parentheses are assumed number of residues. Aminoterminal Fragment D. Only one amino terminus is possible for Fragment residues were determined by dinitrophenylation (1) for porcine E. Values for porcine growth hormone are from Chen et al. (4) growth hormone and Fragments A, B, and C, and by the Edman on the basis of a molecular weight of 22, (18). I Amino acid I Lysine... Histidine... Arginine... Halfcystine.... Aspartic acid.... Threonine Serine Gllltamic acid... Proline.... Glycine... Alanine.... Valine... Methionine... Isoleucine... Leucine Tyrosine... Phenylalanine... Tryptophan... A 4.55 (5) 1.71 (2) 6.84 (7).78 (l)a 9.9 (9) 4.63 (5) 8.35 (8) (17) 4.79 (5) 5.1 (5) 1.41 (1) 5.41 (5).57 (l)d 4.25 (4) (16) 4.22 (4) 7.34 (7) (1 P B 5.17 (5) 3.68 (4).83 (1)o 7.56 (8) 2.66 (3) 2.98 (3) 5.5 (5) 1.1 (1) 3.6 (3) 3.23 (3).91 (1).61 (l)d.78 (1) 8.15 (8) 2.54 (3) 1.93 (2) Fragment C.6 (1) 2.13 (2) 2.1 (2)b 2.13 (2) 1.3 (1) 1.16 (1) 1.17 (1) 1.47 (2) D.97 (1) 1.9 (1) 1.5 (1)d.72 (I) E.92 (1) 1.18 (l)d Porcine growth hormone lf Aminoterminal amino acid.... N.F.Q a As Saminoethylcysteine or Scarboxymethylcysteine. b As cysteic acid. c Not including Fragment E. d As homoserine plus homoserine lactone. e Detected by the Ehrlich reaction (19). f Measured by reaction with ATbromosuccinimide (3, 14). g N.F., not found. Arg LYS Phe Ala Phe, Ala Complete reduction, judged by amino acid analysis, was obtained whether or not a nitrogen barrier was used. For aminoethylation of the reduced protein, the same procedure was used for reduction, and.2 ml of ethyleneimine (1) was added for each 7.5 ml of reduction mixture. The resulting solution was incubated with stirring for 2 hours at room temperature, and the protein was recovered by dialysis and lyophilization. Cleavage with Cyanogen Bromide and Separation of Products Protein was dissolved in 7% formic acid (11) at a concentration of 1 mg per ml. Solid cyanogen bromide (Eastman), equal in weight to the protein, was added, and the mixture was incubated at room temperature for 6 hours. The solution was then diluted with water to 1 to 297, formic acid and lyophilized. The dried product was suspended in water and lyophilized once more. For separation of cleavage products, the treated protein was dissolved in a minimal amount of 7% formic acid; this solution was diluted with water to 2% formic acid and applied to a column of Sephadex G75 in 2% formic acid. A column (3.7 X 17 cm), eluted at a flow rate of 22.5 ml per hour, was used to separate the products from the cleavage of 1 g of protein (Fig. 1). Digestion of Fragment B with Enzymes and Purification of PeptidesFragment B was dissolved in.5 y. NH,HCO, to make a 1 y. solution and digested with 2 y. by weight of Lltosylamido 2phenylethyl chloromethyl ketonetreated trypsin (Worthington) or chymotrypsin (Worthington) for 3 hours at 37. Digestion with pepsin (Sigma) was carried out at room temperature for 2 hours in 5% formic acid with 2% by weight of enzyme. Tryptic peptides were separated by chromatography on Dowex 5X2 or Aminex & 15s resin (BioRad, Richmond, California) under the conditions described by Schroeder et al. (12). Peptides were detected by reaction with 2,4,6trinitrobenzenesulfonic acid (13). Samples of.2 ml were taken from each tube collected at chromatography and were dried in a vacuum over H2S4 and NaOH flakes. The residue was dissolved in 2.5 ml of 2,4,6trinitrobenzenesulfonic acid reagent (4 mg of 2,4,6trinitrobenzenesulfonic acid per 5 ml of 4% borax), and the solution was incubated in the dark for 3 min. After addition of.4 ml of 2 N HCl, the optical density of the solution was measured at 35 mp. Peptides were further purified as necessary by paper electrophoresis at ph 1.9 (acetic acidformic acidwater, 8:2:9, by volume), ph 3.5 (acetic acidpyridinewater, 1: 1:289, by volume), or ph 6.5 (acetic acidpyridinewater, 12:4:36, by volume). Chymotryptic and peptic peptides were isolated and purified only by electrophoresis.

3 Issue of July 1, 197 J. B. Mills, S. C. Howard, S. Scapa, and A. E. Wilhelmi 339 Peptides derived flom tlyptic digestion of Fraglnenl B Values are expressed as mole ratios with assumed number of residues in parentheses. II Amino acid Lysine... Histidine.... Arginine... Halfcystinea.... Aspartic acid... Threonine.... Serine... Homoserine.... Glutamic acid... Proline.... Glycine... Alanine.... Valine... Isoleucine.... Leucine.... Tyrosine.... Phenylalanine... Tl.75 (1 1. (1 1 ) T2 T3 T4 I 1. (l)l.oo (1: 1.6 (I 2.3 (2).89 (1) 2.1 (2).98 (1.99 (1.91 (1.98 (1 1. (1 ) 1 i ; Peptide T5a T5b T6.94 (1) 1.1 (1).98 (1) 1. ( (1) 3.7 (3) 2.8 (2.97 (1) 2.1 (2) 1.2 ( (1) 1.7 (1) 1.18 (1) 1.11 (1.7F (1).74 (1) 1.5 (1).97 (1 _ 1 I) 1 ) ) Tla 1.4 (1.95 (1 1.9 (1.98 (1 1. (1.93 (1 T7b 1.2 (1.99 (1.98 ( (1 T&I 1.11 (1.87 (1 1.1 (1.97 ( (1 2.4 (2.94 (1.94 (1 T8b 1.69 (2) 1.11 (1) 1. (1) 2.16 (2).89 (1) Yield ( %) N.hl.c I.G , Q As Saminoethylcysteine or Scarboxymethylcyst,eine. * Sum of homoserine plus homoserine lactone. c N.M., not measured. d Halfcystine as Scarboxymethylcysteine to.78 Od 1.8 Aminoterminal Analysis and Edman DegradattinAminoterminal amino acids of the intact protein were determined by dinitrophenylation as described by FraenkelConrat, Harris, and Levy (14). The subtractive adaptation of the (15) was used to determine the amino acid sequence of peptides. An appropriate sample of the peptide was dissolved in.2 ml of a mixture of pyridinewatertriethylamine (25 : 24: 1, by volume), 5 ~1 of phenyl isothiocyanate were added, and the solution was incubated at 38 for 2 hours. A few drops of water were added until the solution became milky, and the mixture extracted once with toluene. The lower, aqueous phase was dried in a vacuum at 6 over solid PZOs and NaOH. The residue was dissolved in 2 drops of anhydrous trifluoroacetic acid and incubated at 38 for 3 min. The trifluoroacetic acid was removed under reduced pressure over solid NaOH; the residue, dissolved or suspended in.2 ml of.1 N HCl, was extracted once with toluene. The aqueous layer was dried in a vacuum over HBSOd and solid NaOH. The residue was dissolved in the pyridinewatertriethylamine solvent, an appropriate sample was taken for amino acid analysis, and the remainder was carried through another degradation cycle..2 M sodiumcitrate buffer, ph 2.2, and this solution was applied directly to the amino acid analyzer. Partial acid hydrolysis was carried out in a sealed, evacuated tube in.5 N HCl at 15 for 2 hours. Net Charge of PeptidesThe method used to determine the net charge of peptides is an adaptation of the method described by Offord (16). The mobility of the peptide is determined, relative to aspartic acid, on paper electrophoresis at ph 6.5. The mobility is given a positive sign if the peptide migrates in the same direction as aspartic acid. The net charge is calculated according to the equation, e =.38 mmf, where e is the net charge, m is the mobility relative to aspartic acid, 1/1 is the molecular weight of the peptide determined by amino acid analysis, and.38 is a constant calculated from the mean experimental mobilities of glutamic acid, aspartic acid, lysine, and arginine. This equation is similar to one described by Gray (17) for calculating the net charge of ldimethylaminonaphthalene5sulfonyl peptides. It is not valid for peptides containing histidine because of the uncertainty of the pk, of peptidelinked histidine. Digestion of Peptides with Enzymes and Partial Acid Hydrolysis Isolation of Cyanogen Bromide FragmentsCleavage of the Peptides were digested with trypsin or carboxypeptidase B protein by cyanogen bromide was always at least 96% complete, (Worthington, treated with diisopropyl fluorophosphate) in.5% judged by disappearance of methionine. Fig. 1 shows the elu NH,HCO, at 37. The carboxypeptidase B was found to be tion of the cleavage products from Sephadex G75. Peaks i contaminated with significant carboxypeptidase A activity. and 2 give almost identical amino acid analyses and apparently Aliquots of carboxypeptidase digests were pipetted into 2 ml of represent a single product in varying degrees of aggregation. RESULTS

4 341 Partial of Porcine Growth Hormone Vol. 245, No. 13 Peptides derived from chymotryptic digestion of Fragment B Values are expressed as mole ratios with assumed number of residues in parentheses. Amino acid Lysine... Histidine.... Arginine... Halfcystinea.... Aspartic acid.... Threonine... Serine... Homoserineb... Glutamic acid.... Proline... Glycine... Alanine... Valine.... Isoleucine... Leucine... Tyrosine... Phenylalanine... i. Cl c2 c3.75 (I) 4.14 (4).96 (1) 1. (1) 2.3 (2).88 (1).51 (1) 2.2 (2) 1.14 (1) 1.5 (1) 3.1 (3) 1.1 (1) 2.6 (2) III Peptide c4 C6 c7 C8.82 (1) 3.8 (3).98 (1) 1.8 (1) 1.12 (1) 1.7 (2) 1.2 (1) 3.1 (3).78 (1) 2.6 (2).73 (1) 1.75 (2).92 (1).96 (1) 1.3 (1) 1. (1).75 (1).82 (1) 1.6 (1).93 (1) 2.2 (2) Cl1 Cl2 Cl3 Cl4 Cl5 Cl7 Cl8 Cl9 Lysine... Histidine... Arginine... Halfcystinea... Aspartic acid... Threonine... Serine... Homoserineb.... Glutamic acid... Proline.... Glycine... Alanine.... Valine... Isoleucine... Leucine... Tyrosine... Phenylalanine (2).82 (1) 1. (1).97 (1).9 (1).78 (1) 1.18 (1).9 (1) 1.2 (1) 3.11 (3).95 (1) 1. (1) 1.1 (1) 1.5 (1).82 (1).97 (1) 1.2 (I).88 (1).93 (1).77 (1) 5.26 (5) 1.9 (1) 1.3 (1) 2.9 (3).86 (I).66 (1).71 (1) 1.2 (1) 1.5 (I) a As Saminoethylcysteine. h Homoserine plus homoserine lactone. IV Amino acid sequence of Peptide T2 Values in parentheses are molar ratios. Residues removed in each step of the are in italics.... Step 2. Step3... Step 4. Step 5. SerAspAspAlaLeuLeuLys 1.24 Lys (NAa), Asp (2.5), Ser (.7), Ala (l.oo), Leu (2.2) LYS PA), Asp (1.$9), Ser (.7), Ala (.99), Leu (1.95) LYS (NQ, Asp (.75), Ser (O.lO), Ala (.99), Leu (1.89) LYS (N41, Asp (.69), Ser (.12), Ala (.49), Leu (1.75) LYS (N4, Asp (.62), Ser (.12), Ala (.41), Leu (1.19) a NA, not analyzed.

5 Issue of July 1, 197 J. B. Mills, S. C. Howard, X. Xcapa, and A. E. Wilhelmi 3411 Amino acid sequence of Peptide T4 Values in parentheses are molar ratios. Residues removed in each step of the are in italics., AlaGlyGlnIleLeuLys Step I... LYS (NAa), Glu (l.oo), Gly (l.ol), Ala (O.lZ), Ile (.97), Leu (1.) Step2....._... LYS WA), Glu (1.2), Gly (.62), Ala (.9), Ile (.93), Leu (.98) Step3....._..._..._... Glu (.8), Gly (.41), Ala (O.ll), Ile (.98), Leu (1.3) Step4...._..._..._... LYS WA), Glu (.64), Gly (.26), Ala (.6), Zle (.64), Leu (1.3) Step 5... Lys (O.BSb), Glu (.59), Gly (.22), Ala (.5), Ile (.64), Leu (.68) n NA, not analyzed. b There is often some loss of lysine during the. V Amino acid sequence of Peptide T6 Values in parentheses are molar ratios. Residues removed in each step of the are in italics PheAspThrAsnLeuArg. Step l... Arg (NAa), Asp (1.98), Thr (.94), Leu (l.ol), Phe (.9) Step Arg (N4, Asp (f.7f), Thr (1.1), Leu (l.oo), Phe (.6) Step 3... Arg WA), Asp (1.41), Thr (.58), Leu (1.2), Phe (.6) Carboxnpeptidase 5 miy Arg (l.oo), Asn (O.lO), Leu (.52) GO min. Arg (l.oo), Asn (.61), Leu (1.11) NA, not analyzed. VI VII Amino acid sequence of Peptide T7a Values in parentheses are molar ratios. Residues removed in each step of the are in italics......_. _ Step2....._... Step _..... Step4... AlaGluThrTyrLeuArg Arg (NAQ), Thr (l.oo), Glu (.96), Ala (.8), Leu (1.9), Tyr (.9) Arg WA), Thr (l.oo), Glu (.43), Ala (lost), Leu (1.18), Tyr (.66) Arg (NA), Thr (.51), Glu (.29), Ala (.6), Leu (l.oo), Tyr (.5) Arg (.98), Thr (.41), Glu (.28), Ala (.(i), Leu (1.3), Tyr (.2) NA, not analyzed. This product is called Fragment A. Peak S represents Fragment B, which is not present if native porcine growth hormone is cleaved, but can be generated from Peak 1 or!z by reduction with Pmercaptoethanol. This indicates that Fragments A and B are joined by a disulfide bridge in the intact molecule. Peak 4 represents a mixture of three fragments: C, D, and E. These fragments were purified by paper electrophoresis at ph 3.5. Amino acid analyses of all the fragments are given in Table I. Amino Acid s of Fragments D and ESince each of these peptides was derived from cyanogen bromide cleavage, homoserine must be carboxyterminal. The sequence of Fragment E is AlaHsr.l TWO cycles of the applied to Fragment D removed phenylalanine (95 To) and proline (52 Q, respectively, revealing the sequence of this fragment to be PheProAlaHsr. Amino Acid of Fragment CFragment C contained 1 The abbreviations used are: Hsr, homoserine or homoserine lactone; CmCys, Scarboxymethylcysteine. no homoserine and therefore represents the carboxyterminal portion of the porcine growth hormone molecule. The amino acid sequence of the carboxyterminal octapeptide of porcine growth hormone has already been reported (2). A sample of Fragment C, as the Xcarboxymethylderivative, was subjected to one cycle of the, resulting in the loss (1%) of the single lysine residue. Digestion of Fragment C with carboxypeptidase resulted in the release of phenylalanine only. Digestion of Fragment C with chymotrybsin resulted in the release of two peptides, Ca and Cb. Peptide Ca had an amino acid composition consistent with the sequence ValGlu SerSerCmCysAlaPhe, which is part of the sequence already reported (2). Peptide Cb had the composition (Lys, Argz, CmCys, Phe). Carboxypeptidase A released only phenylalanine, and three cycles of the released, sequentially, lysine (64 %), carboxymethylcysteine (53 %), and arginine (57%). The amino acid sequence of carboxymethylated Fragment C is therefore : LysCmCysArgArgPheVal

6 3412 Partial of Porcine Growth Hormone Vol. 245, Ko. 13 GluSerSerCmCysAlaPhe. This fragment is obtained intact, whether native or reduced porcine growth hormone is cleaved with cyanogen bromide. The 2 halfcystine residues in Fragment C are therefore connected by a disulfide bond in the native hormone. Amino Acid of Fragment BThe amino acid compositions of peptides from tryptic and chymotryptic digestion of Fragment I3 are summarized in Table II and Table III. Since the chymotryptic peptides were isolated only by paper electrophoresis, and the steps necessary for purification varied from one peptide to another, the yields of the peptides were meaningless and therefore are not given. Fragment B was also digested with pepsin, but the resulting peptides were mostly identical with the chymotryptic peptides. Only one, P8, contributed useful data. Its amino acid composition, lysine (.93), aspartic acid (l.ll), glycine (.99), leucine (1.95), and tyrosine (OM), established the association of Peptides C8 and Cl3 (see Table XI). Peptide T1The amino acid sequence, ValHsr, is obvious Aminoacid sequence of Peptide TYb Values in parentheses are molar ratios. Residues removed in each step of the are in italics.. AspLeuHisLys Lys (.99), His (1.2), Asp (O), Leu (.85) Step 2. Lys (.99), His (1.Il), Asp (.4), Leu (.1) Step 3. Lys (l.oo), His (O.ZZ), Asp (O), Leu () VIII from the composition. This peptide represents the carboxyterminal portion of the fragment. Peptide T$The amino acid sequence of this peptide was determined by the (Table IV). The peptide was acidic on paper electrophoresis, and therefore both the aspartic acid residues must exist as the acid and not the amide. Peptide T4The amino acid sequence of this peptide was determined by the (Table V). Although the yields are poor in Steps 2 and 3, as is often the case when glycine and glutamine are aminoterminal, the data are unequivocal. The basic behavior of the peptide on electrophoresis at ph 6.5 and the calculated net charge of +1.4 indicate the presence of one amide group. The presence of glutamine was confirmed by hydrolysis with leucine aminopeptidase. Peptide TSuThe did not remove an amino acid from this peptide, suggesting the possibility of aminoterminal pyrrolidone carboxylic acid with which the net charge of 1.1 is consistent. Peptide TSbFour applications of the did not remove an amino acid from this peptide, a result similar to that with Peptide T5a. Prolonged tryptic digestion of T5b released two peptides with amino acid compositions identical with those of Peptides T5a and T6. Digestion of T5b with carboxypeptidase resulted in the release of arginine, leucine, and asparagine. Peptide TGThis peptide was neutral on electrophoresis at ph 6.5, indicating the presence of one amide group. The Edman degradation and digestion with carboxypeptidase B (contaminated with carboxypeptidase A) led to the sequence shown in Table VI. Amino acid sequence of Peptide T8b Values in parentheses () are molar ratios. Residues removed in each step of the are in italics Carboxypeptidase 2 min hrs Step Step Partial acid hydrolysis Peptide Al Peptide A Step l Step Step Peptide A3.... Peptide B Step Step a NA, not analyzed. ArgGluLeuGluAspGlySerProArg 1.8 No amino acids released Ax (47%) IX Arg (.88), Asp (1.7), Ser (.99), Glu (2.14), Pro (l.lo), Gly (1.2), Leu (1.) Arg (.83), Asp (1.2), Ser (l.oo), Glu (1.36), Pro (.77), Gly (1.4), Leu (.91) Arg (.84), Asp (1.3), Ser (.98), Glu (1_18), Pro (.88), Gly (1.2), Leu (.44) Arg (.97), Asp (1.2), Glu (1.84), Leu (.83) 1.79 Arg (.77)) G111 (2. lo), Leu (1.).97 Arc/ ( 4, Glu (2.18), Leu (1.) Arg (NAG), Glu (J&8), Leu (1.3) Arg (N4, Glu (1.51), Leu (.48) Asp (1.OO) Arg (.73), Ser (1.7), Pro (1.17), Gly (.96) +1.5 Arg (.73), Ser (.99), Pro (1.5), GZy (.16) Arg (.73), Ser (.49), Pro (l.lo), Gly (.22) Arg (.73), Ser (.38), Pro (.44), Gly (.24)

7 Issue of July 1, 197 J. B. Mills, X. C. Howard, 8. Xcapa, and A. E. Wilhelmi 3413 Studies on chymotryptic peptides from Fragment B Values in parentheses are molar ratios. Residues removed in each step of the are in ifalics. Peptide C5 ArgGluLeuGl~~AspProSerGlyArgAlaGlyGlnIleLeu 7 T8b, T Step 2 Peptide C8 GlyLeuLeu Gly (O.SO), Leu (2.) Peptide Cl3 LysAsnTyr Lys (), Asp (l.ol), Tyr (.99) St,ep 2 Lys () Asp (.46), Tyr (1.) Peptide Cl4 LysLysAspLeuHisLysAlaGluThrTyr Step 2 Step 3 Step 4 Peptide Cl5 LysGlnThrTyr 1.15 Step 2 Lys Lys (.1), (.14), Thr Thr (.96), (l.ol), Glu Glu (1.5), (.46), Tyr Tyr (.81) (.83), Peptide Cl9 SerAeCysaPhe n AeCys (.69), Ser (.15), Phe (1.) Step 2 AeCys (.23), Ser (.21), Phe (1.) a AeCys, Saminoethylcysteine. X Arg (.87), Asp (1.7), Ser (1.9), Glu (3.2), Pro (.78), Gly (2.6), Ala (l.ol), Ile (.84), Leu (1.76) Arg (.83), Asp (l.ll), Ser (1.8), Glu (6.5/t), Pro (.84), Gly (2.23), Ala (.98), Ile (.85), Lell (1.86) Lys (1.&Y), His (.95), Asp (1.7), Thr (.93), Glu (1.12), Ala (1.4), Leu (1.7), Tyr (.81) Lys (1.12), His (.99), Asp (.82), Thr (.99), Glu (1.22), Ala (1.6), Leu (1.7), Tyr (1.1) Lys (1.18), His (,96), Asp (.63), Thr (1.8), Glu (1.14), Ala (1.4), Leu (.96), Tyr (.97) Lys (.92), His (1.7), Asp (.56), Thr (1.9), Glu (1.37), Ala (1.2), Leu (.56) Tyr (.97) Peptide T7aThe peptide was neutral and therefore contained no amide groups. Its amino acid sequence was determined by four steps of the (Table VII). Peptide T7bThe amino acid sequence of this peptide was determined by three cycles of the (Table VIII). Digestion with leucine aminopeptidase released aspartic acid, but no asparagine. The net charge of +.78 indicated that the histidine residue was almost completely charged at ph 6.5. Peptide T8aThe peptide, in the Xcarboxymethyl form, was neutral at ph 6.5, and therefore the single aspartic acid residue was present as asparagine. Two steps of the Edman degradation removed asparagine (47%) and tyrosine (55%) sequentially. Peptide T&The net charge of 1.8 indicated the presence of no amide groups. The amino acid sequence was determined by, carboxypeptidase digestion, and partial acid hydrolysis (Table IX). Chymotryptic PeptidesStructural studies were done on six of the chymotryptic peptides; the data are summarized in Table X. In other cases, the composition of the peptides was sufficient to establish, without ambiguity, overlapping portions of tryptic peptides. The complete amino acid sequence of Fragment B is shown in italics in Table XI. The peptides contributing to the elucidation of the structure are shown beneath the corresponding sequence in the fragment. DISCUSSION Porcine growth hormone contains 3 methionine residues; four fragments should be produced by cleavage with cyanogen bromide. We have isolated five such fragments and have tentatively concluded that Fragments D and E are each derived from a heterogeneous aminoterminal region of the protein, similar to that in ox growth hormone (2, 21). This interpretation is consistent with our aminoterminal analysis of the protein using dinitrofluorobenzene, in which some preparations yielded aminoterminal alanine (4), and with the aminoterminal sequence, PheProAlaMetProLeu, reported by Papkoff, Li, and Liu (22). It has been impossible to determine accurately the relative yields of Fragments D and E due to their multiple forms (homo

8 3414 Partial of Porcine Growth Homone Vol. 245, No. 13 Amino acid sequence of Fragment B The sequence, in italics, is shown above the tryptic, chy theses was established only by composition and comparison motryptic, and peptic peptides used in determining the struc with other peptides. The numbering of residues refers only ture of Fragment B. The position of residues separated by to Fragment B and has no relationship with their positions in short dashes was established by chemical analysis, while that intact porcine growth hormone. of residues shown separated by commas and enclosed in paren XI T8b T4 c5 Cl5 T5b T5a T6 T2 (Cl5) Cl7 c4 c3 c7 c2 Cl Cl3 P ArgGluLeuGluAspGlySerProArgAlaGlyGlnIleLeuLys ArgGluLeuGluAspGlySerProArg AlaGlyGlnIleLeuLys ArgGlu(Leu, Glu,Asp, Gly, Ser,Pro,Arg,Ala, Gly, Gln, Ile,Leu) Lys GlnThrTyrAspLysPheAspThrAsnLeuArgSerAspAspAlaLeuLeuLys (Gln,Thr,Tyr,Asp,Lys,Phe,Asp,Thr)AsnLeuArg (Gln,Thr,Tyr,Asp,Lys) PheAspThrAsnLeuArg SerAspAspAlaLeuLeuLys GinThrTyr (Asp,Lys, Phe, Asp,Thr, Asn,Leu, Arg, Ser,Asp,Asp, Ala,Leu,Leu) (Asp,Lys,Phe,Asp,Thr,Asn,Leu) (Arg,Ser,Asp,Asp,Ala,Leu,Leu) (AsP,LYs,P~~) (Asp,Thr,Asn,Leu) (Asp,Thr,Asn,Leu,Arg,Ser,Asp,Asp,Ala,Leu,Leu) Lys (LYS, T8a T3 T7b T7a Tl (Cl3) (W C8 Cl9 Cl1 C6 Cl4 Cl8 Cl AsnTyrGlyLeuLeuSerCysPheLysLysAspLeuHisLysAlaGluThrTyrLeuArgValHsr AsnTyr(Gly,Leu,Leu,Ser,Cys,Phe,Lys) AsnTyr,Asn,Tyr,Gly,Leu,Leu) GlyLeuLeu SerCysPhe LYS AspLeuHisLys AlaGluThrTyrLeuArg (Lys,Lys,Asp,Leu,His) (Lys,Ala,Glu,Thr,Tyr) LysLysAspLeu(His,Lys,Ala,Glu,Thr,Tyr) ValHsr (Leu,Arg,Val,Hsr) (Arg,Val,Hsr) Comparison of carboxyterminal regions of several species of growth hormone The dash in the human sequence represents an amino acid deletion. Ox., LysCysArgArgPheGlyGluAlaSerCys AlaPheCOOH Porcine. LysCysArgArgPheValGluSerSerCys AlaPheCOOH Human GlnCysArg SerValGluGlySerCys GlyPheCOOH XII that of horse growth hormone (23). It is different in only 2 residues from the sequence of the comparable region of ox growth hormone (24). The same region of human growth hormone (1) is 1 residue shorter and is different from pig growth hormone in 4 other residues. These comparisons are summarized in Table XII. The amino acid sequence of Fragment B shows a high degree of homology with Residues 124 to 177 in human growth hormone (1). This sequence of 55 amino acids in the porcine hormone is 1 residue longer than that of the human, and there are 16 other amino acid substitutions. Preliminary evidence, in the form of methionine diagonal experiments (25), indicates that Fragment B is adjacent to Fragment C (the carboxyterminal fragment) in native porcine growth hormone. Assuming this to be so, there is at least 67% homology between human and pig growth hormones in the carboxyterminal third of the molecules. serine and homoserine lactone) and to their incomplete elution from paper during purification. The amino acid sequence of the carboxyterminal dodecapeptide of porcine growth hormone (Fragment C) is identical with

9 Issue of July 1, 197 J. B. Mills, S. C. Howard, X. Xcapa, and A. E. Wilhelmi 3415 We have been unable to detect any evidence for an amino 9. terminal pentapeptide fragment, as reported (2, 21) to be present 1. in ox growth hormone. Likewise we have found no microheterogeneity in Peptide T8b, which corresponds to Peptides 11. Tpl,2 of Fellows and Rogol (2), showing partial substitution of valine for leucine AcknowledgmentsWe thank Misses M. Yager, M. L. Willcox, and Mrs. L. Chang for their invaluable assistance REFERENCES LI, C. H., LIU, W. K., AND DIXON, J. S., Arch. Biochem. Biophys., 133, 7 (1969). FIZLLOWS, R. E., JR., BND ROGOL, A. D., J. Biol. Chem., 244, (1969). DELLACHA, J. M., SANTOM~, J. A., AND PALADINI, A. C., Ann. 19. N. Y. Acad. Sci., 148, 313 (1968). CHEN, H.C., WILHELMI, A. E., AND HOWARD, S. C., J. Biol. Chem., 246, 342 (197). 2. P~RLOW, A. F., WILHELIIZI, A. E., AND REICHERT, L. E., JR., 21. Enclocrinology, 77, 1126 (1965). 22. BENSON, J. V., AND PATTERSON, J. A., Anal. Chem., 37, 118 (1965). RAMACHANDRAN, L. K., AND WITKOP, B., J. Amer. Chem.. i$oc., , 428 (1959). PETERS, T., JR.,.C. R. Trav. Lab. Cadsberg, 31,227 (1959) CRESTFIELD, A. M., MOORE, S., AND STEIN, W. H., J. Biol. Chem., 238, 622 (1963). RAFTERY, M. A., AND COLE, R. D., Biochem. Biophys. Res. Commun., 1, 467 (1963). STEERS, E., JR., CRAVEN, G. R., ANFINSEN, C. B., AND BE THUNE, J. L., J. Biol. Chem., 24,2478 (1965). SCHROEDER, W. A., JONES, R. T., CORMICR, J., AND MCCALLA, K., Anal. Chem., 34, 157 (1962). SATAKE, K., AND OKUYAMA, T., J. Biochem. (Tokyo), 47, 454 (196). FRAENKELCONR~T, H., HARRIS, J. I., AND LEVY, A. L., Methods Biochem. Anal., 2,359 (1955). EDMAN, P., Acta Chem. &and., 4, 283 (195). OFFORD, R. E., Nature, 211,591 (1966). GRAY, W. R., in C. H.W. HIRS (Editor), Methods in enzymology, Vol. XI, Academic Press, New York, 1967, p ELLIS, G. J., MARLER, E., CIIEN, H. C., AND WILHELMI, A. E., Fed. Proc., 26, 348 (1966). BEALE, D., in H. LEHMANN AND R. G. HUNTSMAN (editors), Man s haemoglobins, J. B. Lippincott Company, Philadelphia, 1966, p MILLS, J. B., Nature, 213, 631 (1967). WALLIS, M., Fed. Eur. Biochem. Sot. Lett., 3, 118 (1969). PAPKOFF, H., LI, C. H., AND LIU, W. K., Arch. Biochem. Biophys., 96,216 (1962). OLIVER, L., AND HARTREE, A. S., Biochem. J., 19,19 (1968). SANTOMI~, J. A., WOLFF,NSTEIN, C. E. M., BISCOGLIO, M., AND PALADINI, A. C., Arch. Biochem. Biophys., 116,19 (1966). TANG, J., AND HARTLEY, B. S., Biochem. J., 12,593 (1967).